Project Summary/Abstract Acute myeloid leukemia (AML) is a challenging disease to cure. The only known ?cure? currently is hematopoietic stem cell transplantation (HSCT) which requires conditioning prior to transplantation. The population that AML affects does not tolerate myeloablative conditioning well so a reduced intensity conditioning is used instead. However, the relapse rate is unacceptably high in patients that receive reduced intensity conditioning. Immunotherapy is a promising new field for treatment of all cancer types and we aim to improve immunotherapy for AML patients. The benefits of targeting NK cells lie within its inherent tumor surveillance ability. Adaptive NK cells, which are expanded after cytomegalovirus (CMV) reactivation, are a subtype of NK cells that display enhanced killing and cytokine production. Adaptive NK cells are considered adaptive because of their memory-like phenotype, showing enhanced activation following re-exposure. Adaptive NK cells are also associated with lower relapse following HSCT. Adaptive NK cells are easily identified as CD57+NKG2C+. The function of NKG2C has not been thoroughly defined on adaptive NK cells. NKG2C binds to HLA-E, a non- classical major histocompatibility complex (MHC) class I molecule, with peptide specificity to activate NK cells. NKG2C+ cells also downregulate NKG2A, a family member of NKG2C that also binds to HLA-E with peptide specificity but inhibits NK cells. Our proposal aims to exploit NKG2C on adaptive NK cells for tumor immunotherapy. In Aim 1, we look at the role of NKG2C specifically on adaptive NK cells. We will use a cell line, K562, that lack MHC class I molecules resulting in potent NK cell activation due to lack of inhibition. We will manipulate these cells to express HLA-E (K562-E) and with these cells to determine whether there is a signaling function through NKG2C that is unique to adaptive NK cells or whether it is the decrease in NKG2A expression and subsequent reduction of inhibition in adaptive NK cells that leads to their enhanced function. We can use peripheral blood NK cells to study the function of NKG2C on adaptive NK cells, but the number of adaptive NK cells in humans is variable. In Aim 2, we will use induced pluripotent stem cell (iPSC) derived NK cells (iNK) with transduced NKG2C to kill tumor cells. We will specifically target tumor cells using a Trispecific Killer Engager that activates NKG2C on the iNK, linking them with tumor target while simultaneously stimulating NKG2C and IL15 receptor. Aim 2 creates a specific translational therapy that can be scaled for clinical trials. Overall, this project aims to exploit NKG2C as a functional receptor for immunotherapy.